1. Field of the Invention
The present invention relates to a confocal laser scanning microscope apparatus, which is suitable for a planar determinate quantity measurement, etc., for example, of ion concentration of a cell, its image recording method, and a recording medium on which its program is recorded.
2. Description of the Related Art
Conventionally, a confocal laser scanning microscope apparatus that narrows laser light from a light source into micro spotlight with an objective lens, scans an observation specimen with the spotlight, converts light from the observation specimen, such as transmitted light, reflected light, fluorescence, etc. into an electric signal with a photoelectric converter such as a photomultiplier, etc., and displays an observation image on an image monitor is known as a scanning microscope apparatus.
In the meantime, a method quantifying intracellular free ion concentration with an ion sensitivity indicator has become rapidly widespread as a study method in neuroscience and cell biology fields in recent years. By way of example, with a measurement of intracellular calcium ion concentration, a fluorescence measurement using a fluorescence dye fura-2, which is a calcium ion sensitivity indicator, for a sample cell is made. With the fluorescence measurement at this time, after a particular region of a cell is excited with two different wavelengths (340 nm and 380 nm), and a stimulus substance (Bradykinin) is given, quantification is made by obtaining a ratio of respective fluorescence intensities, and a change in the intensities is measured with a fluorescence spectrophotometer, observed with a microscope, or the like.
Additionally, Japanese Patent No. 2749069 proposes a fluorescence microscope apparatus that measures calcium ion concentration with the above described fluorescence dye fura-2. With this fluorescence microscope apparatus, only light having a wavelength transmitted by a switched filter among lights from a light source is irradiated on a sample by arbitrarily switching a plurality of interference filters whose transmission wavelengths are different, and a sample image is obtained by capturing and image-processing the light from the sample, so that a measurement using a plurality of lights having different wavelengths as excitation lights, like a 2-wavelength excitation 1-wavelength fluorescence measurement using the fluorescence dye fura-2, or the like can be made.
However, the above described proposal discloses not a configuration in the case where a measurement is made with a confocal laser scanning microscope apparatus, but only a configuration in the case where a normal optical microscope apparatus that optically obtains a two-dimensional image is used.
Additionally, no proposals are conventionally made as to a configuration in the case where a measurement using a plurality of lights having different wavelengths as excitation lights like a 2-wavelength excitation 1-wavelength fluorescence measurement, etc., which is applied to a calcium ion concentration measurement using the above described fluorescence dye fura-2, a hydrogen ion concentration measurement, a macromolecule concentration measurement, etc., is made with the use of a confocal laser scanning microscope apparatus.